Comparison of Mediterranean fruit fly (Ceratitis capitata) (Tephritidae) bisexual and genetic sexing strains: development, evaluation and economics

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1 Proceedings of 6th International Fruit Fly Symposium 6 10 May 2002, Stellenbosch, South Africa pp Comparison of Mediterranean fruit fly (Ceratitis capitata) (Tephritidae) bisexual and genetic sexing strains: development, evaluation and economics C. Caceres 1, J.P. Cayol 2, W. Enkerlin 2 *, G. Franz 1, J. Hendrichs 2 & A.S. Robinson 1 1 Entomology Unit, Agriculture and Biotechnology Laboratory, Agency s Laboratories, A-2444 Seibersdorf, Austria 2 Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA, Wagramerstrasse 5, A-1400, Vienna, Austria In Medfly, Ceratitis capitata, sterile insect technique (SIT) programmes, the use of genetic sexing strains (GSS) is now routine. The use of these strains in mass-rearing facilities enables them to produce only males for irradiation and release. The advantages of using these strains are described together with genetic approaches used for their construction. The early use of the strains in massrearing facilities highlighted important limitations that were not apparent during their construction and small-scale evaluation. Using a combination of new genetic and rearing approaches the problems have essentially been solved. The potential use of one GSS in many different geographic locations raised concerns about mating compatibility. A very detailed field-cage study using populations from very diverse areas showed that these strains had no unexpected impact on mating compatibility. The economics of the use of GSS in SIT programmes is described in detail, covering both their mass production and use in the field. The analysis reveals a much improved cost/benefit ratio when GSS are used. Future improvements that will further enhance the use of GSS are also discussed. BACKGROUND The sterile insect technique (SIT) is an established technology for the suppression and/or eradication of selected key insect pests of man, his livestock and crops (Tan 2000). The technology involves the mass production and release, on an area-wide basis,of large numbers of sexually sterile insects into a field population. The released males mate with the females in the field resulting in no production of offspring. Following repeated releases of the sterilized insects, the field population is suppressed and in certain circumstances eradication can be achieved (Hendrichs 2000). To be effective,sit does not require the release of sterile females as they do not contribute to the transfer of sterility to the wild population. Thus, mass production of sterile females is unnecessary and the technique requires that only sterile male insects are released (Knipling 1955; Robinson et al. 1999; McInnis et al. 1994; Franz & McInnis 1995; Hendrichs et al. 1995). In some cases the release of females can have a negative impact. As a result of this situation, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture initiated activities in 1981 to develop special strains for the Medfly, Ceratitis capitata, that could be used to produce only males for areawide SIT programmes. These activities included research and development at the FAO/IAEA *To whom correspondence should be addressed. w.r.enkerlin@iaea.org Laboratories at Seibersdorf in Austria, and the funding of three Coordinated Research Projects (CRPs) (IAEA 1990, 1997; Genetica 2002). With the completion of the final CRP, almost all of the Medfly rearing facilities worldwide are using these genetic sexing strains (GSS) for their SIT programmes (Robinson et al. 1999; Table 1). Prior to the start of the first CRP very little was known about the genetics of Medfly, and the task of this CRP was to develop many of the essential genetic tools, e.g. mutations, polytene chromosome analysis and male-linked translocations, which are essential for the construction of a GSS. Table 1. Medfly mass-rearing facilities using GSS with the potential capacity in millions of males/week. Country Strain Capacity Argentina SEIB 6 (VIENNA 7/Tol) 80 Australia VIENNA 7/Mix 7 Chile SEIB 6 (VIENNA 7) 40 Greece (Crete) SEIB 7/Mix 3 Guatemala VIENNA 7/Tol 1600 Hawaii (CDFA) VIENNA 7/Tol 100 Peru VIENNA 7/Mix 100 Portugal (Madeira) VIENNA 7/Mix 50 Seibersdorf VIENNA 7-D53/Mix 25 South Africa VIENNA 7-D53/Mix 7 Hawaii VIENNA 7/Tol 200 (USDA/APHIS) Total 2212

2 368 Proceedings of the 6th International Fruit Fly Symposium At the conclusion of the second CRP, the first transfer of a GSS to an operational SIT programme in Guatemala took place. Studies carried out during the second CRP demonstrated the efficacy of an all-male release in the field (McInnis et al. 1994) and included information on a temperature-sensitive mutation, tsl, in Medfly (Franz et al. 1996). The use of this mutation has been central to the practical application of GSS in Medfly. Some of the most serious problems to be confronted in the use of GSS in operational programmes concerned the stability of the strains under mass-rearing conditions. These have now been essentially solved by a combination of new rearing methods and the use of more appropriate translocations. Initially it was assumed that the sex determination mechanism of Medfly would be very similar to that of Drosophila melanogaster. However, it is now known that in most tephritid fruit flies there is probably a single gene that initiates male sex determination (Zapater & Robinson 1986; Willhoeft & Franz 1996) in contrast to D. melanogaster, where a chromosomal balance system operates. The use of polytene chromosome analysis (Kerremans et al. 1990, 1992; Zacharopoulou et al. 1991) has been essential for the development of stable GSS, by enabling both mutations and translocation breakpoints to be accurately mapped (Franz & Kerremans 1993; Franz et al. 1994; Kerremans & Franz 1994, 1995). DEVELOPMENT, IMPROVEMENTS AND REARING OF MEDFLY GENETIC SEXING STRAINS FOR USE IN SIT Benefits of genetic sexing strains (GSS) The benefits of using GSS for Medfly SIT have been articulated many times and were summarized by Hendrichs et al. (1995) as follows: a. economic savings in rearing, irradiation, packaging, transport and release; b. increased male quality as male pupae can be irradiated 24 h before emergence, instead of 48 h when also irradiating females; c. several-fold increase in field effectiveness as the sterile sperm is not wasted in matings with sterile females, and sterile males compete better for wild females. Some studies also showed that sterile males disperse more in the absence of sterile females; d. in the absence of sterile females in the fly emergence containers, sterile males can be held longer (without mating taking place with sterile females) and thus released at a more mature age, thereby reducing losses before males reach full sexual capacity; e. simplified and more precise monitoring activities when using female attractants, as the recapture of sterile males is largely reduced, thus also significantly reducing the risk of mis-identification (plus the value of not removing many valuable sterile males); f. no damage in certain types of fruit due to absence of oviposition stings by sterile females, and reduced transfer of pathogenic fungi and bacteria to such fruit; g. increased applicability of SIT for Medfly suppression in fruit growing regions, because the reduced cost and absence of oviposition damage to the fruit enables the routine use of sterile males as a biological insecticide to replace chemical bait-sprays during fruiting seasons; h. increased bio-safety, as an accidental release of non-irradiated flies would only include males, and escaping females from mass-rearing would have reduced fitness. This is particularly important for mass-rearing facilities located in fruit fly-free areas or areas where Medfly eradication is the objective. In conclusion, the most important benefit is the increased efficiency of SIT, as it has been shown in many Medfly studies that male-only releases introduce 3 4 times more sterility into the target population than do bi-sexual releases (McInnis et al. 1986; Robinson et al. 1986; McInnis et al. 1994; Rendon et al. 2000). Choice of sexing system The practicality and economics of GSS use depends on the choice of the appropriate selectable marker in the sexing system. Initially the white pupae (wp) mutation (Rössler 1979) was used in combination with seed sorters to separate white (female) and brown (male) pupae. However, it became clear that this type of selectable marker has two significant disadvantages. First of all, the sex separation can be achieved only after the costly mass-rearing step, i.e. expensive diet has to be wasted on rearing females and, in addition, the females had to be killed and disposed of in a safe way. Second, the use of seed sorters is not easy. Such machines are very expensive, especially considering the separation capacity needed in most mass-rearing facilities; they are relatively complicated and are not very accurate (c. 5% contamination with females in the male-only

3 Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 369 product and a significant loss of male pupae). Furthermore, the sorting process can damage the pupae, resulting in flies with reduced flight ability. Acknowledging these severe disadvantages led to the development of an alternative sexing system. This is based on a temperature-sensitive lethal (tsl) mutation that allows females to be killed as early as the egg stage by applying elevated temperatures. This is, in mass-rearing, the earliest and most practical stage (as large numbers of eggs are maintained for two days in plastic bottles) and the killing of the females requires only very simple and cheap equipment (a water bath). By now, extensive experience with the tsl-based GSS is available and an accuracy of 99.5% or better can be achieved routinely even in very large mass-rearing operations (Fisher 1998, 2000). The problem of stability The first mass-rearing of Medfly genetic sexing strains revealed that stability was going to be a major problem that would need to be solved before these strains could be transferred to operational SIT programmes (Robinson & van Heemert 1982; Robinson 1984; Hooper et al. 1986). Solving the problem involved two strategies, 1) inducing new strains with inherently improved genetic characteristics (Franz et al. 1994) and 2) developing new rearing procedures (Fisher 1998, 2000; Fisher & Caceres 2000). By combining these strategies the problem of stability has essentially been brought under control. During the first large-scale mass-rearing of these strains, novel biological events occurred which also impacted on stability. These unexpected genetic problems had also to be solved. In addition to controlling stability there has been a continuous improvement in the quality control profile of GSS. The solutions Improved strains Strain improvement has been achieved by: very accurate cytological mapping of the markers used in the strains i.e. white pupae and tsl, making it possible to select translocations with breakpoints very close to the markers. This had the immediate effect of improving stability significantly, reducing recombination by c. 65%; very accurate cytological mapping of the position of the male determining gene on the Y chromosome and by analysing the position of the translocation breakpoint on that chromosome made it possible to significantly reduce the impact of a particular genetic destabilizing event. There is now a clear understanding of the genetic factors that are involved in the stability of genetic sexing strains in Medfly, and using this information their stability has been improved to a level enabling them to be mass-reared in a predictable and reliable way (Caceres et al. 2000). Inversions are now being introduced into GSS with the aim of further increasing stability and facilitating the introduction of different genetic backgrounds, if specifically required. In addition, a phenotypic marker is being evaluated which may remove uncertainties regarding identification of released flies in the field. However, when tens or hundreds of millions of insects are being reared weekly, random biological events can still occur and cause problems that can only be solved by introduction of large changes in the rearing methods. Improved rearing strategies Instability occurs during mass-rearing of GSS following rare genetic recombination events. Occasionally, some of the different phenotypes, produced as a result of these events, accumulate because of a higher fitness in the colony, as traditional mass-rearing strategy returns these individuals back into the colony. In order to prevent this accumulation, the Filter Rearing System (FRS) was developed (Fisher & Caceres 2000). In this system, (Fig. 1), a small colony of the GSS is kept under relaxed rearing conditions and is checked at every generation for the presence of unexpected flies which, if found, are discarded. Eggs from this colony are used to produce a large colony following a small number of amplification steps. Eggs from this large colony are then used to produce males for release. No insects that have been through the mass-rearing procedure are returned to the small initial colony. Production is essentially a one-way street. The FRS has been successful in a number of large-scale facilities that mass-rear tsl-gss and has demonstrated its effectiveness in maintaining strain stability for many generations. The use of the FRS has another important function in relation to the overall quality of the mass-reared insects, both in GSS but also in bi-sexual strains (BSS). It is possible to design an FRS in order to try to maintain conditions that are closer to the natural environment of the flies.

4 370 Proceedings of the 6th International Fruit Fly Symposium Fig. 1. Schematic representation for the operation of a filter rearing system (FRS) to maintain stability in a GSS. Such a relaxed rearing environment for the small initial colony may reduce strain deterioration (e.g. less inbreeding as a result of a strong inadvertent selection of laboratory adapted traits that are not well fitted for field conditions), thereby extending the viable life of a colonized strain under mass-rearing conditions. Improved quality control profiles The quality control profile of GSS can be negatively influenced by the survival of genetically unbalanced individuals to different stages in the developmental cycle. This problem is linked to the genetic structure of the GSS and is associated with the 50% sterility that has been characteristic of such strains. In earlier versions of GSS this sterility manifested itself at a relatively late stage of development, such that a significant proportion of the pupae produced were genetically unbalanced, resulting in low adult emergence and in reduced adult quality for this type of genetically unbalanced pupae. This led to overall reduced quality control values for emergence, flight ability and mating performance. Selecting specific translocations for GSS has solved this problem. The current GSS, VIENNA 7 and VIENNA 8, were not only chosen because of their increased stability but also because their genetic structure is such that no, or only very few, genetically unbalanced individuals survive beyond egg/early larval stage. From that stage onwards only the desired flies are reared resulting in qual-

5 Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 371 ity control values similar to standard BSS. These strains show a clearly better performance in massrearing than older versions of GSS. A problem that needs to be addressed GSS based on temperature sensitivity and translocations require a colony at least three times as large as a BSS colony to produce the same number of eggs (see below). This is due to the fact that 1) the strains are semi-sterile due to the translocation,and half the eggs are killed as a result of the temperature treatment, and 2) female Medflies of GSS are homozygous for the tsl mutation and show a reduced viability. Inducing a particular type of translocation that will not be associated with semi-sterility could solve the first problem. Making some changes to the way adult colonies are maintained may help solve the second. This could involve changes in cage design and improved climatic/environmental controls. The size of the colony is currently the major disadvantage to the use of GSS involving the tsl mutation. Even so, as described below in the sections on evaluation of competitiveness and economics, the benefits of using these GSS in operational Medfly SIT programmes significantly outweigh the disadvantages during rearing. EVALUATION OF SEXUAL BEHAVIOUR, COMPETITIVENESS AND COMPATIBILITY OF MEDFLY GENETIC SEXING STRAINS In terms of the sexual behaviour and mating compatibility of GSS there are two issues that have been repeatedly raised in relation to the potential use of these strains in operational SIT programmes. The first concerns the genetic background of GSS. This often differs from that of the target Medfly population, raising the question of the mating compatibility among the different Medfly populations in the world. The second is the fact that GSS carry mutations in the form of selectable genes and translocations, the impact of which, on behaviour, is unknown. In view of these two considerations and the increasing demand for Medfly GSS for SIT suppression programmes, these strains had to undergo thorough and systematic testing for sexual compatibility and mating competitiveness with the target wild population before they could be cleared for use in SIT programmes. In fact, very few insect mass-reared strains, if any, have been as intensely and thoroughly tested as Medfly GSS, especially tsl strains. The most relevant work done on the assessment of the sexual behaviour, competitiveness and mating compatibility of Medfly GSS is reviewed below. First, however, we will review the information available on the sexual and other behaviours of Medflies in the wild or in the laboratory, against which to assess GSS behaviour. Baseline information on Medfly sexual behaviour There is a solid base of information on mating competitiveness and sexual behaviour of wild and laboratory BSS against which the performance of GSS can be measured. Concerns about the sexual behaviour and competitiveness of mass-reared and laboratory Medfly strains were raised in the 1970s. As a first step, Holbrook & Fujimoto (1970) assessed the mating competitiveness of irradiated and non-irradiated Medflies. Rössler (1975) and Wong & Nakahara (1978) measured the ability of laboratory-reared Medfly males to inseminate females, in comparison with wild flies. Fried (1971), Boller & Chambers (1977), Boller et al. (1981), Chambers et al. (1983), Boller & Calkins (1984) and Orozco et al. (1983) published a collection of quality control tests for fruit flies and a quality control manual for Medfly mass-rearing facilities. Prokopy & Hendrichs (1979) provided a description of the lek mating behaviour of wild Medflies and Zapien et al. (1983) carried out the first Medfly quality control test on a field-caged host tree to assess wild female mate choice for competing wild and sterile males. This was followed by studies in the open field to confirm the observed Medfly behaviours under natural conditions (Hendrichs & Hendrichs 1990; Hendrichs et al. 1991; Whittier et al. 1992, 1994, Shelly & Whittier 1996). These observations were used to compare the behaviour of mass-reared insects with wild insects (Chambers et al. 1983; Orozco et al. 1983; Zapien et al. 1983; Calkins et al. 1994). In the s, the various effects of mass-rearing (Calkins & Ashley 1989; Calkins 1991; Calkins et al. 1996), nutritional status (Blay & Yuval 1997; Bravo & Zucoloto 1998), and irradiation (Wong et al. 1982, 1983) on the sexual competitiveness and mating behaviour (Liimatainen et al. 1997), including female mating-induced changes (Jang et al. 1998), were studied. Comparative assessments of the mating competitiveness of various BSS were also done, most notably in countries using this type of strain for large-scale SIT programmes, such as Mexico (Liedo et al. 1996). All of these studies concluded that flies from standard BSS were less competitive in mating than their

6 372 Proceedings of the 6th International Fruit Fly Symposium wild counterparts, that some sequences of courtship behaviour were shortened (Briceño & Eberhard 1998), and that these changes appeared to be mainly due to the mass-rearing conditions, the irradiation process and the age or number of generations of the strain under mass-rearing conditions (Eberhard 2000). That the age of the strain has severe implications was also demonstrated in an extreme case in Hawaii,where the use of one particular BSS (HiLab) in mass-rearing for over 40 years without refreshment resulted in behavioural incompatibility between wild and mass-reared flies (McInnis et al. 1996a). This represents the only case of incompatibility documented in Medfly and the problem was solved with a change of strain, emphasizing the need to renew BSS often as is being done routinely in most SIT programmes. In 1998, on the basis of all the above studies, an international manual was developed to harmonize quality control procedures, including the fieldcage mating test (FAO/IAEA/USDA 2003). In view of the female-choice mating system in Medfly, the field-cage mating test is probably the most important component of quality control assessment in this species. Carried out under semi-natural conditions and in the presence of wild females, this field-cage mating test allows the detection of deviations from the standard value of mating performance expected from any mass-reared strain. Worldwide assessment of mating compatibility among Medfly populations In the early days of Medfly SIT, strains were colonized from the local wild population, increased in numbers, sterilized, and released in the same general area. In the early 90s, the increased transboundary shipment of sterile Medflies and the availability of the first Medfly GSS for large-scale use in SIT programmes, resulted in the same strain being used in different countries, regions or even continents. As these strains are based on genetic material that often differs in the geographical origin from that of the target wild population, concern was raised regarding their sexual compatibility and competitiveness at other locations. From 1994 to 1999, within the framework of an FAO/IAEA Coordinated Research Project, the mating compatibility of Medflies from wild and laboratory- or mass-reared BSS and GSS from various origins was compared. This resulted in a comparative assessment of wild flies from nine countries representing five continents, five different GSS (four tsl-based and one wp-based) and six laboratory or mass-reared BSS under field-cage and/or laboratory conditions. The assessment of each strain s mating compatibility and competitiveness (Cayol 2000a), together with the detailed slow-motion video analysis of their courtship behaviour (Lux et al. 2002; Cayol et al. 2002) concluded that no major differences could be found among the strains, regardless of their genetic background. The only exception was Medfly females from Madeira Island (Portugal), which were only partially compatible with males from VIENNA 7-97 (Pereira unpubl. data; Cayol unpubl. data). It was concluded that BSS and GSS, regardless of their origin, could be used against different Medfly populations, as well as for outbreaks of unknown origin as in the cases of California and Florida in the U.S.A. However, it was again confirmed that long-term mass-rearing and irradiation procedures reduce the mating competitiveness and shorten the courtship duration of laboratory-reared flies (Briceño & Eberhard 1998; Cayol 2000b; Eberhard 2000). Assessment of the behaviour of Medfly GSS First generation GSS: white pupae (wp) strains Robinson et al. (1986) assessed the mating competitiveness and sexual compatibility of a GSS based on wp with wild flies from Procida Island, Italy, and Hooper et al. (1986) assessed the behaviour of this strain under mass-rearing conditions. The effectiveness of this wp GSS was successfully assessed in a large open-field trial in Israel in 1989/90 (Nitzan et al. 1993), resulting in only 0.4% of fruits with live maggots in a 500 ha guava test area. A similar strain was also tested in southern Tunisia (Cayol & Zaraï 1999) and resulted in Medfly being successfully suppressed in some oases. An improved version of the original wp GSS, SEIB 6-96, was tested under field-cage conditions in Argentina (Cayol et al. 1999). This strain was used for several years in Mendoza and various provinces of Patagonia, where it has been very successful in controlling Medfly population (De Longo et al. 2000). These studies, together with the comparative assessment of the detailed courtship behaviour of wild flies (Cayol 2000a; Lux et al. 2002), concluded that there were no major differences between the wild flies and the GSS flies. Consequently, the use of wp GSS represented a major step forward, even though it still involved the

7 Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 373 production and mechanical separation of females at the pupal stage. Second generation GSS: temperature-sensitive lethal (tsl) strains The first tsl-gss was made available in the early 1990s (Franz et al. 1994). This represented a major breakthrough and an increase in cost-effectiveness of Medfly SIT since it allowed sex separation at the egg-stage. In view of the fact that this type of strain carries two mutations, wp and tsl, in addition to the translocation, it was thoroughly tested with respect to sexual behaviour, competitiveness, and field effectiveness. The original VIENNA 42, and its improved successors, have been tested at all levels in the laboratory, in field cages and in the open field. Hendrichs et al. (1993) tested the dispersal and survival of VIENNA 42 in citrus orchards in Greece and showed that these flies dispersed and survived as well as those from a BSS. The mating competitiveness of this strain was then tested under fieldcage conditions against wild flies from Greece (Hendrichs et al. 1996). Again, tsl males proved to be as competitive as males of a BSS, although less competitive than wild flies. The VIENNA 43/44 tsl was then tested in comparison with a wp GSS over nearly 300 ha in southern Tunisia for 11 months (Cayol et al. 1997; Cayol & Zaraï 1999). Both GSS were found to be equally competitive and their use in Tunisia resulted in the successful suppression of the resident Medfly population. The VIENNA 4/Tol-94 tsl was then developed based on a local genetic background from Guatemala. The performance of this strain was first monitored under mass-rearing conditions (Caceres et al. 2000) and then the behaviour in the laboratory, in field cages in Guatemala (Rendon et al. 1996a), in greenhouses in Vienna (Cayol et al. 2002), and in the open field (Rendon et al. 1996a,b; McInnis et al. 1996b). The relative mating competitiveness of VIENNA 4/Tol-94, VIENNA 42 and two standard BSS (HiLab and Petapa) was assessed in field-cage experiments in Hawaii and in Guatemala (Lance et al. 2000). The authors reported that even though quantitative differences could be found between wild and laboratory flies, no major qualitative differences were found between BSS and GSS. Most importantly, VIENNA 4/Tol-94 was also compared against the standard Petapa BSS, during three consecutive years in a large open field trial in Guatemala, involving routine operational SIT activities over large areas (Rendon et al. 2000). This comparison showed that the tsl GSS was three to five times more effective at inducing egg sterility in the field than was the BSS. Kaspi & Yuval (2000) measured how adult protein-feeding increased the mating competitiveness of tsl GSS males from Guatemala. McInnis et al. (2002) compared the remating frequency of this tsl GSS with that of BSS under field-cage conditions in Guatemala. The authors did not find any significant differences between BSS and tsl GSS. Mass-reared males (either BSS or GSS) had a similar reduction in mating compared to their wild counterparts. After confirming mating compatibility, tsl GSS pupae are being shipped weekly from the El Pino facility in Guatemala to Israel and Jordan, and have been proved successful in suppressing the Medfly population in the Arava/Araba Valley south of the Dead Sea (Rössler et al. 2000). From the end of 1999 and following preliminary feasibility and mating compatibility studies (Barnes & Eyles 2000), pupae from the same tsl GSS have been shipped from Guatemala to be released for routine Medfly suppression over the Hex River Valley of the Western Cape Province of South Africa. This has resulted in the elimination of insecticide used for Medfly suppression and a halving in the number of table grape boxes rejected for export due to the presence of Medfly larvae (B.N. Barnes, unpubl. data). VIENNA 7-D53/Mix-2001, which was very recently made available by the FAO/IAEA Laboratories in Seibersdorf, represents one of the latest developments of tsl Medfly strains. Compared to its predecessor tsl strains, the genetic stability of VIENNA 7-D53/Mix-2001 has been improved by the addition of an inversion, and the strain is based on genetic material of a mixed wild-type strain. VIENNA 7-D53/Mix-2001 has already been tested successfully in field cages against wild flies in Israel (Gazit & Rössler 2001) where it has been as competitive as VIENNA 4/Tol-94, achieving about one third of wild female mates when competing with wild males. From November 2001, the weekly releases of eight million tsl GSS from Guatemala is being supplemented with an additional five million pupae of this new strain from the FAO/IAEA Laboratories in Austria. Because of their genetic composition and their wide use in large-scale SIT programmes in various countries and continents, the Medfly GSS, and especially the tsl strains, have been thoroughly tested. To date, much more is known on behaviour, field competitiveness, genetics and morphometrics of GSS strains than what was known for

8 374 Proceedings of the 6th International Fruit Fly Symposium BSS used in large SIT programmes for many years. It can be concluded that there is strong evidence that different Medfly populations in the world have not evolved mating incompatibilities, with the possible exception of the population on Madeira Island. Furthermore, there is no evidence that the GSS available today behave any differently than BSS, and both types of strains are affected equally by mass-rearing, irradiation and excessive longterm rearing. ECONOMIC COMPARISON OF THE USE OF MEDFLY BISEXUAL AND GENETIC SEXING STRAINS Medfly GSS, in particular the tsl-based GSS, have been increasingly used in large operational SIT projects since the 1990s. Thus, sufficient information on routine costs is available to make some general comparisons of the economics of their use compared with that of standard BSS. Intuitively, the production of male-only tslbased GSS would appear to be more economical compared to the cost of producing males and females in the case of BSS. The fact, however, is that producing only males of tsl-based GSS is currently slightly more expensive because overall, the operational and capital costs of male-only tsl-gss rearing facilities are greater than that of the normal BSS rearing facilities. Nevertheless, when the costs of all programme operations are included in the estimates, it becomes clear that the utilization of tsl-based GSS is much more cost-effective than using BSS. Rearing process For comparisons of the two production systems (i.e. tsl-based GSS and BSS) the production process has to be separated into two major parts: 1) adult colony and 2) larval and pupal rearing. While for the tsl-gss the proportion of costs between the colony and larval rearing and pupal handling is approximately 1:4 (20% colony; 80% larval and pupal rearing), for the BSS the proportion is 1:9 (10% colony and 90% larval and pupal rearing). When comparing the two rearing systems, the major cost differences in rearing procedures are observed in the adult colony (egg production). Although for the larval and pupal rearing there are also differences in some of the basic requirements, such as the amount of diet and space, the differences in the two systems compensate each other as will be explained in the following sections. Since maintenance of the adult colony, compared to the larval and pupal rearing, represents the smallest proportion of the overall production, the difference in cost between the two systems is no more than 15%. Adult colony Compared to the rearing of BSS, different procedures are required for tsl-based GSS due to the genetic composition of such strains (Caceres et al. 2000). For a tsl-based GSS a large adult colony is required because 50% of all eggs produced are rendered unviable by the partial genetic sterility of GSS males. Of the remaining eggs, 50% represent the temperature-sensitive females and these are killed by the temperature treatment. As a consequence, only 25% of all eggs produced will result in males for release. An additional reduction in the overall egg production per cage is caused by the sensitivity of the tsl females to stresses such as high densities in the cages. These factors together result in a three-fold increase in the space required for the adult colony rooms, a sixfold increase in number of cages and three-fold increase in labour (Table 2). An increase in space brings an increase in the basic utilities required such as water and power, as well as a cost increase in maintenance and depreciation of the additional equipment and infrastructure. Larval and pupal rearing For the maintenance of a tsl-based GSS colony, larval trays are seeded at half the egg density used for a BSS. This has to be done to protect the temperature-sensitive females against the high temperatures resulting from larval overcrowding. In addition, the trays have to be kept for four additional days due to the lower temperatures and the resultant slower female development rate. This results in a four-fold increase in diet use and a five-fold increase in space required for larval production for colony maintenance, compared to the larval production for a BSS colony. The increased amount of diet required to maintain the breeding colony for the tsl-based GSS system is compensated by the 20% less diet required for rearing the male larvae when compared to the amount required for rearing both males and females for BSS field releases (Table 2). Comparing the overall volume of diet required to rear either a tsl-based GSS or a BSS, both systems are more or less equal (Table 2). Although the savings in diet for adult release in the case of a GSS are small in absolute numbers (20%), they affect the major part of diet use, i.e. for larval rearing for release. Here, 2.6-fold more diet is used than in the larval

9 Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 375 Table 2. Comparison of rearing requirements between a tsl-gss and a BSS to produce 100 million flying Medfly males/week. Requirements Ratio Number/quantity BSS:GSS BSS GSS Adult colony Colony size (million of adults) 1.0: Colony replacement (millions of pupae) 1.0: No. cages for male only production 1.0: Egging room (m 2 ); male only 1.0: No. cages for colony maintenance 1: Egging room (m 2 ); colony 1: Adult diet (sugar) (kg/day) 1.0: Adult diet (yeast hydrolysate) (kg/day) 1.0: Workers (colony + filter) 1:3 3 9 Larval rearing/pupal handling Workers 1: Larval diet/day (kg) for colony 1.0: Larval diet/day (kg) for release 1.0: Subtotal 1.0: Rearing area for colony (m 2 ) 1.0: Rearing area for release (m 2 ) 1.0: Subtotal 1.0: Rearing area (total) 1.0: Total pupal production/week (millions) 1.0: Millions of flying males/week 1: rearing for colony maintenance. The rearing of a tsl-based GSS requires more diet for colony rearing but this represents the smaller proportion of the overall diet use. These relationships virtually lead to equal diet consumption for both systems. In terms of space requirements a similar relationship holds true. The tsl-gss system requires five times as much space for rearing the larvae and pupae that will maintain the breeding colony. However, in contrast, the amount of space required for rearing of the male larvae for adult release is nearly twice as large for the BSS. This is due to a more synchronized development of male-only larvae that allows retrieval of all the larvae in one-and-a-half days compared to the BSS rearing system that requires three days. As in the case of the diet, if the space required for the two systems is summed, the comparison between GSS and BSS comes close to a 1:1 ratio (Table 2). Given that the space for larval and pupal rearing is very similar for both systems, and considering that the type of activities required are in general also very similar, we can infer that the basic utilities (i.e. water, power, etc.) and maintenance costs are practically the same. In summary, for the larval and pupal rearing process for release, which accounts for the largest proportion of the production costs, there is a significant difference in costs between the two rearing systems. At the end, the tsl-based GSS system is currently slightly more expensive with respect to colony maintenance. Overall, the current cost per million tsl-males is not more than 10 15% higher than for the BSS system. However, a new tsl-gss has recently been developed and tested at the Seibersdorf Laboratory, in which mortality of genetically unbalanced individuals occurs much earlier during development. As a result the amount of larval diet required for this strain for male-only larval rearing is not 0.2 but at least 0.4 times lower than the amount required for rearing BSS. This and other recent improvements in GSS rearing are discussed below. Post-production process There are a number of direct and indirect costs of using the BSS that are not incurred when using the tsl-based GSS, and these are transformed into benefits for the programme when a tsl-based GSS is used.the major sources of direct cost reduc-

10 376 Proceedings of the 6th International Fruit Fly Symposium Table 3. Comparison of cost ratios between the use of a BSS and a tsl-gss. Cost Item BSS:GSS Direct costs Rearing 1.0:1.2 Sterilization 1.0:0.5 Shipping 1.0:0.5 Holding and emergence 1.0:0.5 Release 1.0:0.5 Indirect costs Male competitiveness 1.0:0.3 Sterile:fertile fly identification 1.0:0.5 False ID of released sterile flies 1.0:0.5 Outbreak following escape of 1:0 non-irradiated adults Quality loss due to female stings 1:0 tion are related to irradiation, packing, shipping, emerging, feeding, holding, collecting and release. This is due to the fact that for tsl-based GSS only half of the pupae and adult fly volume is handled throughout the process from irradiation and packing to release, compared to BSS where pupae of both sexes have to be irradiated, transported, fed, handled and released. When transforming these cost factors into ratios, the differences between the tsl-based GSS and BSS systems become obvious (Table 3). In addition, there are several sources of indirect costs which have to be taken into account (Table 3). These indirect costs in favour of tsl-based GSS are: 1) significantly improved competitiveness of sterile males in the absence of sterile females (Robinson et al. 1986; McInnis et al. 1994; Rendon et al. 2000); 2) cost savings in the identification of trapped flies to discriminate between sterile and fertile individuals; and 3) more sensitive survey systems based on the use of specific female lures result in significantly reduced recapture of sterile males. In addition, there are other more intangible and indirect benefits of using tsl-based GSS such as: 4) avoiding the false identification of unmarked released sterile females, representing savings when not responding to false outbreaks; 5) significantly increased safety in the event of an escape of non-irradiated adults into pest-free areas, as a result of the non-viability of tsl females; and 6) avoidance of quality loss in some fruit commodities due to the absence of fruit stinging by sterile females. Overall economic advantage of using tsl-gss in SIT operations Using as a hypothetical case the shipment from Guatemala and release of sterile males in Panama, and transforming the above ratios into actual costs in US$/treated km 2 /week, the cost for the tsl-gss was estimated at US$40/km 2 /week, compared with US$ 146/km 2 /week for the BSS, a 3.5-fold cost reduction (Table 4). This difference could be even greater if the other cost factors shown in Table 4 (the non-quantified indirect costs) were added. A range of costs/km 2 /week, including those estimated for the two sexing systems, was entered in a fruit fly economic model (FAO/IAEA and Centre for Environmental Technology Imperial College of Science, Technology and Medicine 2001). The model estimates a number of economic indices for the utilization of the tsl-gss and BSS.The economic indices were estimated by computing the costs and benefits of a Medfly area-wide integrated control programme, again using Medfly control in Panama as an example, where the use of sterile insects would be a significant component. The costs and benefits were transformed into net present value and were projected over a 15-year time frame. Results indicate that when using the tsl-gss system instead of a BSS,the initial project investment is paid one year earlier. The benefit:cost ratio of the programme when using the tsl-gss is 4.9:1 compared to 3.7:1 when the BSS is used (Fig. 2). Furthermore, the net revenues for the fruit industry in the country would be greater by an amount of US$19 million over 15 years if a tsl-gss was used, which is a significant difference for a small fruit industry. This example demonstrates the economic advantages of using a Medfly tsl-gss. These advantages would apply to any situation where the improved Medfly strain is used.mm In conclusion, governments and modern fruit industries are increasingly aware not only of the trade and environmental benefits of using SIT but also the economic benefits. The development of tsl-gss has already helped reduce the costs of area-wide application of SIT against Medfly to the point where SIT is now economically viable for use as a biological insecticide for routine Medfly suppression, rather than to be applied only for eradication or barrier purposes. There are already viable suppression projects in progress in various locations, including South Africa (Barnes et al. 2001) and Israel (Nitzan et al. 1993; Rössler et al. 2000). The utilization of the SIT will continue to

11 Caceres et al.: Medfly bisexual and genetic sexing strains: development, evaluation and economics 377 Table 4. Comparative cost per km 2 per week in US$ for the BSS and the tsl-gss using current approximate costs from Central American SIT operations. Cost Item BSS GSS Direct costs Sterile males Shipping 8 4 Holding and emergence Release Subtotal Indirect costs Male competitiveness Female sterile:fertile identification Subtotal Non-quantified indirect costs False ID of released sterile flies * * Outbreak as a result of escape of non-irradiated adults * * Quality loss due to sterile female stings Unknown Unknown Total Assuming a release density of 1000 males per ha or per square kilometre. 2 Refers to induction of sterility in the field, which is increased three fold by the release of males only (calculated as two additional times direct costs). *Not significant on a km 2 per week basis. Fig. 2. Economic returns for SIT when using a GSS strain (tsl ) and a normal bisexual strain (BSS). increase as the cost-effectiveness of existing SIT technology continues to be optimized. ONGOING IMPROVEMENTS AND FUTURE PROSPECTS A number of recent research findings indicate that the 3.5-fold reduction of Medfly SIT cost/km 2 / week that is achieved when using GSS rather than BSS (Table 4), can be even greater once the following improvements are introduced: 1) Improved productivity Increasing the egg seeding density on larval diet due to early mortality of genetically unbalanced individuals as explained above, and modifying the egging cages used for the release stream, can potentially increase egg production times. 2) Improved use of the Filter Rearing System The FRS can be used to introduce a more relaxed or natural rearing environments to reduce strain deterioration. This would enable the viable life of a colonized strain to be extended under mass-rearing conditions, thus requiring strain renewals less often. 3) Development of protocols for long distance shipment of Medfly eggs Improved productivity as described above may allow facilities to achieve increased economies of scale and invest in increased egg production capacities in order to ship the excess eggs to facilities in other locations. These facilities could produce tsl males for sterilization and release without the need to maintain filter, amplification and release stream colonies. 4) Use of a morphological marker Sergeant (Sr 2 ) is a dominant marker that produces an extra stripe on the abdomen of the adult fly. It can be introduced into a GSS so that males will show the phenotype while females will be wild type at this locus. Initial field tests have not revealed any reduction in the mating competitiveness of flies carrying this marker, and it is now being introduced into a GSS for further evaluation under massrearing conditions. Releasing sterile males that carry this marker would facilitate the sterile:fertile identification process and would

12 378 Proceedings of the 6th International Fruit Fly Symposium Table 5. Comparison of rearing requirements between a tsl-gss and BSS to produce 100 million flying Medfly males per week assuming that the improvements in the GSS rearing system have been incorporated to the production process. Requirements Ratio Number/quantity BSS:GSS BSS GSS Adult colony Colony size (million of adults) 1.0: Colony replacement (millions of pupae) 1.0: No. cages for male only production 1.0: Egging room (m 2 ) 1.0: No. cages for colony maintenance 1.0: Egging room (m 2 ) 1.0: Adult diet (sugar) (kg/day) 1.0: Adults diet (yeast hydrolysate) (kg/day) 1.0: Workers (colony + filter) 1:3 3 9 Larval rearing/pupal handling Workers 1: Larval diet/day (kg) for colony 1.0: Larval diet/day (kg) for release 1.0: Subtotal 1.0: Rearing area (larvae+pupae) for colony (m 2 ) 1.0: Rearing area (larvae+pupae) for release (m 2 ) 1.0: Subtotal 1.0: Rearing area (total) 1.0: Total pupal production/week (millions) 1.0: Millions of flying males per week 1: provide greater confidence in the identification, thus saving substantial amounts of money in unnecessary control and regulatory actions. Furthermore, such a marker would improve the quality of the sterile released insects since no dye would be necessary to mark the sterile insects before release. 5) A new GSS A new strain is being evaluated which has a greatly improved quality control profile probably due to the properties of the translocation used in the strain. Transferring this new GSS to mass-rearing facilities and incorporating the new operational procedures to large-scale mass-rearing will have immediate economic implications. The cost per million tsl-males will be at least equal to, but probably lower than BSS production due to the fact that the space for larval and pupal rearing would be 30% less than for BSS. In addition, the required volume of diet would be 40% less than for BSS (Table 5). Under these conditions it can be inferred that, as larval rearing activities represent around 80% of the total rearing cost, rearing of this new GSS could be at least 10% more economical than a BSS. In comparison with a BSS, use of the new GSS rearing system could reduce the costs of SIT per square kilometer even further than the current estimate of 3.5- to 4.2-fold less expensive. This further reduction in operational costs would make the economic returns of SIT application even more favourable. REFERENCES BARNES, B.N. & EYLES, D.K Feasibility of eradicating Ceratitis spp. fruit flies from South Africa by SIT In: Tan, K.H. (Ed.) Area-wide Management of Fruit Flies and Other Insect Pests. Universiti Sains Malaysia Press, Penang, Malaysia. BARNES, B.N., EYLES, D.K. & SPIES, A Fruit fly control with the sterile insect technique How high will it fly? In: Olckers, T. & Brothers, D.J. (Eds) Proceedings of the 13th Entomological Congress. Entomological Society of Southern Africa, Pretoria, South Africa. BLAY, S. & YUVAL, B Nutritional correlates of reproductive success of male Mediterranean fruit flies (Diptera: Tephritidae). Animal Behaviour 54: BOLLER, E.F. & CALKINS, C Measuring, monitoring and improving the quality of mass-reared Mediterranean fruit flies, Ceratitis capitata Wied. 3. Improve-

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